Identification and characterization of a novel Delta6-fatty acid desaturase gene from Rhizopus arrhizus

It is known that mammalian rpS3 functions as a DNA repair endonuclease and ribosomal protein S3. It was also observed that several ribosomal proteins or DNA repair enzymes are related to apoptosis. We report here a third function of rpS3, induction of apoptosis. The localization of green fluorescent protein (GFP)-rpS3 is changed to the nuclear membrane when lymphocytic cells undergo rpS3-induced apoptosis. Transient expression of GFP-rpS3 activates caspase-8/caspase-3 and sensitizes cytokine-induced apoptosis. Deletion analysis reveals that the two functions of rpS3, DNA repair and apoptosis, use independent functional domains.     

Fatty acid biosynthesis in eukaryotic photosynthetic microalgae: Identification of a microsomal delta 12 desaturase in Chlamydomonas reinhardtii

Polyunsaturated fatty acids (PUFAs) are important components of infant and adult nutrition because they serve as structural elements of cell membranes. Fatty acid desaturases are responsible for the insertion of double bonds into pre-formed fatty acid chains in reactions that require oxygen and reducing equivalents. In this study, the genome-wide characterization of the fatty acid desaturases from seven eukaryotic photosynthetic microalgae was undertaken according to the conserved histidine-rich motifs and phylogenetic profiles. Analysis of these genomes provided insight into the origin and evolution of the pathway of fatty acid biosynthesis in eukaryotic plants. In addition, the candidate enzyme from Chlamydomonas reinhardtii with the highest similarity to the microsomal delta 12 desaturase of Chlorella vulgaris was isolated, and its function was verified by heterologous expression in yeast (Saccharomyces cerevisiae).     

Secretion of pro- and mature Rhizopus arrhizus lipases by Pichia pastoris and properties of the proteins

The lipases of Rhizopus spp. share a high 1,3-regiospecificity toward triacylglycerols, which makes them important enzymes in lipid modification. In the present study, the extracellularly active production of recombinant Rhizopus arrhizus lipase was carried out with genes encoding the mature region (mRAL) and the mRAL having the prosequence (ProRAL) in Pichia pastoris. Two transformed P. pastoris clones containing the multicopy of mRAL and ProRAL genes were separately selected for the production of recombinant enzymes. In a fed-batch cultivation, where methanol feeding was controlled by an on-line methanol analyzer, the supernatant contained 91 mg/L recombinant pro-form lipase (rProRAL) and 80 mg/L recombinant mature lipase (rRAL) after 92 h of cultivation. rProRAL and rRAL were purified by ultrafiltration, SP-Sepharose Rast Flow chromatography, and Butyl-Sepharose Fast Flow chromatography. Molecular weights of rProRAL and rRAL are 32 kDa and 29 kDa, respectively. The amino-terminal analysis showed that the 32-kDa protein was mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL). The specific lipase activities of mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL) and rRAL were 1543 U/mg and 2437 U/mg. The rPro28RAL was more stable than rRAL at pH 4.0–7.0, whereas rRAL was more stable at pH 7.0–10.0. The rPro28RAL had the highest lipase activity toward tributyrin (C₄), whereas rRAL had the highest lipase activity toward tricaprylin (C₈).     

Delta 9-fatty acid desaturase from arachidonic acid-producing fungus. Unique gene sequence and its heterologous expression in a fungus, Aspergillus.

Based on the sequence information for delta 9-desaturase genes (from rat, mouse and yeast), which are involved in the desaturation of palmitic acid and stearic acid to palmitoleic acid and oleic acid, respectively, the corresponding cDNA and genomic gene were cloned from the fungal strain, Mortierella alpina 1S-4, which industrially produces arachidonic acid. There was a cytochrome b5-like domain linked to the carboxyl terminus of this Mortierella desaturase, as also seen in the yeast delta 9-desaturase. The Mortierella delta 9-desaturase genomic gene had only one intron, in which a novel phenomenon was observed: there was a GC-end at the 5'-terminus instead of a GT-end that is, in general, found in introns of eukaryotic genes. The full-length cDNA clone was expressed under the control of an amyB promoter in a filamentous fungus, Aspergillus oryzae, resulting in drastic changes in the fatty acid composition in the transformant cells; the contents of palmitoleic acid (16:1) and oleic acid (18:1) increased significantly, with accompanying decreases in palmitic acid (16:0) and stearic acid (18:0). These changes were controlled by the addition of maltose as a carbon source to the medium. Also, the expression of the gene caused a significant change in the lipid composition in the Aspergillus transformant. Genomic Southern blot analysis of the transformant with the Mortierella delta 9-desaturase gene as a probe confirmed the integration of this gene into the genome of A. oryzae.     

Influence of carbohydrates and polyols on l-lactic acid production and fatty acid formation by Rhizopus arrhizus

Growth and l-lactic acid production on 24 different carbohydrates and polyols (glycerol, mannitol and sorbitol) by Rhizopus arrhizus CCM 8109 were determined. The d- but not the l-forms of xylose, fructose, galactose, mannose, glucose, cellobiose, maltose and sucrose and partially hydrolysed starch were converted to l-lactic acid. Changes in lipid formation and fatty acid composition were detected in biomass grown on the different sugars. In the presence of polyols, growth and considerable production of lipids were observed with little or no lactate production. Invertase was mainly associated with the mycelium during growth on sucrose, whereas glucoamylase and -amylase were produced extracellularly during growth on starch.The authors are with the Department of Biochemical Technology, Faculty of Chemical Technology, Slovak Polytechnical University, Radlinského 9, SK-812 37 Bratislava, Slovak Republic     

Production of lactic acid and byproducts from waste potato starch by <Emphasis Type="Italic">Rhizopus arrhizus</Emphasis>: role of nitrogen sources

Summary Lactic acid was produced by Rhizopus arrhizus using waste potato starch as the substrate. The aim of this study was to identify the role of nitrogen sources and their impact on the formation of lactic acid and associated byproducts. Ammonium sulphate, ammonium nitrate, urea, yeast extract and peptone were assessed in conjunction with various ratios of carbon to nitrogen (C:N). Fermentation media with a low C:N ratio enhanced the production of lactic acid, biomass and ethanol, while a high C:N ratio favoured the production of fumaric acid. Ammonium nitrate appeared to be the most suitable nitrogen source for achieving a high and stable lactic acid yield, and minimizing the production of byproducts such as biomass and ethanol, while urea proved to be the least favourable nitrogen source. Yeast extract and peptone appeared to improve fungal cell growth. The kinetics data revealed that a high concentration of ammonium nitrate enhanced the lactic acid productivity. The maximum lactic acid concentration of 36.4 g/l, representing a yield of 91%, was obtained with addition of 0.909 g/l ammonium nitrate in 32 h.     

大豆硬脂酰-ACP Δ^9脱氢酶(GmSAD)基因家族的鉴定及功能分析

硬脂酰-ACPΔ~9脱氢酶(Stearoyl-acyl carrier proteinΔ~9 desaturase,SAD)在质体中催化单不饱和油酸或棕榈油酸的合成,是控制植物细胞饱和脂肪酸与不饱和脂肪酸比例的关键酶。为解析大豆油酸合成积累调控机制,文中对大豆Glycine max GmSAD家族成员进行全基因组鉴定和保守功能域及理化性质等分析。应用qRT-PCR检测GmSAD各成员的时空表达谱,构建表达载体并通过农杆菌介导烟草Nicotiana tabacum瞬时表达和油酸缺陷型酵母Saccharomyces cerevisiae突变株BY4389遗传转化测试GmSAD酶活性和生物学功能。结果表明,大豆基因组含有5个GmSADs家族成员,其编码酶蛋白均具有二铁中心和SAD酶特有的2个保守组氨酸富集基序(EENRHG和DEKRHE),预测其活性酶蛋白为同源二聚体。系统进化分析显示5个GmSAD分成2个亚组,分别与拟南芥AtSSI2和AtSAD6亲缘关系较近。GmSAD各成员在大豆根、茎、叶、花和不同发育时期种子等组织中表达谱差异明显,其中GmSAD5在发育种子中、晚期高量表达,与油脂富集时期相吻合。烟草叶片瞬时表达GmSAD5可使叶片组织中油酸和总油脂含量分别提高5.56%和2.73%,而硬脂酸含量相应降低2.46%。缺陷型酵母遗传转化测试显示,过表达GmSAD5能恢复缺陷酵母合成单不饱和油酸的能力和促进油脂积累。总之,大豆GmSAD5对硬脂酸底物选择性较强,能高效催化单不饱和油酸的生物合成,为大豆种子油酸和总油脂积累机制的研究奠定了基础,也可作为油脂品质遗传改良的优异靶标。     

Molecular cloning and expression analysis of a delta 6-fatty acid desaturase gene from Rhizopus stolonifer strain YF6 which can accumulate high levels of gamma-linolenic acid

The delta 6-desaturase gene was cloned from Rhizopus stolonifer, which could accumulate up to 49% of gamma-linolenic acid (GLA, C18:3 Δ^6,9,12) to the total fatty acids. The cloned DNA contains a 1,380 bp open reading frame encoding a protein of 460 amino acids, which showed high similarity to those of fungal delta 6-desaturases with three conserved histidine-rich motifs and HPGG motif. Notably, this deduced sequence had a shorter C-terminus. Results demonstrated that the cDNA sequence exhibited delta 6-desaturase activity by accumulation of about 22.4 % of GLA to the total fatty acids in the recombinant Pichia pastoris strain GS115.     

Construction of a <Emphasis Type="Italic">Rhizopus arrhizus</Emphasis> glucoamylase gene suitable for expression in distinct host: introns spliced artificially by PCR

Glucoamylase is an industrially extremely important enzyme in the fermentative production of ethanol, used in the enzymatic conversion of starch into high glucose and fructose syrups. The aim of this study is to construct a Rhizopus arrhizus glucoamylase gene (RaGA)—introns artificially spliced by PCR—suitable for expression in S. cerevisiae host and tried expressing in Picha pastoris. In previous work, we failed in amplifying glucoamylase gene from R. arrhizus by RT-PCR, so several primers were designed to splicing the introns by PCR in vitro. Sequence analysis shown that all introns in the RaGA were deleted correctly and no mutant was induced in the extrons compared with the RaGA gene originally cloned. The RaGA gene artificially constructed was transferred into P. pastoris integrative expression vectors pPIC9 (containing а-factor). Consequently, the plasmids pPIC9-RaGA was lineared by SacI and inserted into P. pastoris GS115 (His⁻) genome downstream of the 5′AOX1 promoter by the method of electroporation. Induction by 0.75% methanol for 72 h led to synthesis of secreted glucoamylase. So it is demonstrated that the glucoamylase gene has been expressed in and secreted from P. pastoris.     

Probing the mechanism of a cyanobacterial Δ9 fatty acid desaturase from Spirulina platensis C1 (Arthrospira sp. PCC 9438)

The initial and rate determining step in the mechanism of fatty acid desaturases has been proposed to be breakage of one of the C---H bonds at the site of the incipient double bond. This has been investigated and supported for a number of eukaryotic fatty acid desaturases through the use of kinetic isotope effect experiments with deuterated substrates. In order to probe the reaction catalyzed by the cyanobacterial Δ9 desaturase and compare it to the eukaryotic desaturases, the desC gene of Spirulina platensis, strain C1 (Arthrospira sp. PCC 9438) was expressed in a desaturase mutant of baker's yeast. Kinetic isotope effects were performed by culturing yeast transformants with deuterated thia-substituted stearic acids. A large kinetic isotope effect was found for the 9 position, in qualitative agreement with results from eukaryotic desaturases.     

Functional characterization of a cotton late embryogenesis-abundant D113 gene promoter in transgenic tobacco

Previous studies have shown that mRNA and protein encoded by late embryogenesis-abundant (LEA) gene D113 from Gossypium hirsutum L. accumulate at high levels in mature seeds and also in response to abscisic acid (ABA) in young embryo. In this study, we studied the expression of four promoter 5′ deletion constructs (−1383, −974, −578 and −158) of the LEA D113 gene fused to beta-glucuronidase (GUS). GUS activity analysis revealed that the −578 promoter fragment was necessary to direct seed-specific GUS expression in transgenic tobacco plants (Nicotiana tabacum L.). To further investigate the expression pattern of LEA D113 promoter under environmental stresses, 2-week-old transgenic tobacco seedlings were exposed to ABA, dehydration, high salinity and cold treatments. GUS activity in the seedlings was quantified fluorimetrically, and expression was also observed by histochemical staining. An apparent increase in GUS activity was found in plants harboring constructs −1383, −974 and −578 after 24 h of ABA or high-salinity treatments, as well as after 10 days of dehydration. By contrast, only a slight increase was observed in all the three lines after cold treatment. Virtually no change in expression was found in construct −158 in response to dehydration, salinity and cold, but there was a moderate response to ABA, suggesting that the region between −574 and −158 was necessary for dehydration- and salinity-dependent expression, whereas ABA-responsive cis-acting elements might be located in the −158 region of the promoter.     

Identification of a novel bifunctional delta12/delta15 fatty acid desaturase from a basidiomycete, Coprinus cinereus TD#822-2

A new gene encoding a delta12 fatty acid desaturase-related protein was cloned from a multicellular basidiomycete Coprinus cinereus TD#822-2. The 1326 bp full-length gene, designated as Cop-odeA, codes for a putative protein of 442 amino acids with a MW of 49224. The Cop-odeA yeast transformant accumulated four new fatty acids identified as 9,12-hexadecadienoic acid, 9,12,15-hexadecatrienoic acid, linoleic acid, and alpha-linolenic acid, which comprised 8.8%, 1.0%, 29.0%, and 0.6% of the total fatty acids, respectively. The Cop-odeA protein was confirmed to be a novel bifunctional fatty acid desaturase with both high delta12 desaturase activity and unusual delta15 desaturase activity.     

Isolation of a maize beta-glucosidase gene promoter and characterization of its activity in transgenic tobacco

The β-glucosidase gene of maize (ZmGLU1) was suggested to hydrolyze cytokinin-conjugate and release free cytokinin during plant growth and development. A clone containing the upstream region of ZmGLU1 was isolated and sequenced from a maize genomic library. The full-length ZmGLU1 promoter and a series of its 5′ deletions were fused to the beta-glucuronidase (GUS) reporter gene and transferred into tobacco. The GUS activity of transgenic plants was assayed at various developmental stages. The results showed that ZmGLU1 promoter-driven GUS gene had the highest expression level in the roots and that the expression of GUS gene declined during seed maturation and down to the lowest level in mature seeds. The ZmGLU1 promoter-driven GUS expression increased during seed germination, reaching a peak on day 11. The results also showed that this promoter could be inhibited by 6-BA, trans-zeatin, and NAA, but was not affected by GA₃, ABA, SA, cold, salt, drought, and submergence treatments. The histochemical staining revealed that GUS activity was located in vigorous cell division zones with dominant staining associated with vascular tissues. Deletion analysis showed that the promoter contained a putative leaf-specific and stem-specific negative regulative element and two putative enhancers.     

Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization

The sequence corresponding to the mature lipase of Rhizopus oryzae WPG (ROLw) was subcloned in the pPIC9K expression vector, with a strong AOX₁ promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. The His-tagged lipase was expressed in Pichia Pastoris X₃₃ and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni-NTA resin). High level expression of the lipase by Pichia Pastoris X₃₃ cells harbouring the lipase gene containing expression vector was observed upon induction with 2.5 g/l methanol at 28°C; the specific activity of the purified His₆-ROLw was 1,500 or 760 U/mg using olive oil emulsion or tributyrin as substrates, respectively. To check the importance of Asn 134 His substitution in the affinity and substrate selectivity of ROLw, the mutant His₆-ROLw-N134H was overexpressed in Pichia Pastoris X33 and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged ROLw-N134H was 5,900 and 35 U/mg using olive oil emulsion or tributyrin as substrate. A comparative study of the wild type (His₆-ROLw) and the mutant (His₆-ROLw-N134H) proteins was carried out. A 3D structure model of ROLw was built using the RNL structure as template. We have concluded that a slight increase in the exposed hydrophilic residues on the surface of ROLw as compared to RNL (ROLwN134H) could be responsible for a higher selectivity of ROlw for long and short chain triacylglycerols at the lipid/water interface and then explaining the importance of Asn 134 for the chain length specificity of ROLw. This property is quite rare among Rhizopus lipases and gives this new lipase great potential for use in the field of biocatalysis.